Production techniques for monoclonal antibodies generally fall into two categories: growth in ascites fluid and culture of immortalized cells secreting the antibodies in fermenters or cell culture chambers of various designs. For growth in cell culture, it is generally desirable to supply many nutrients in the form of animal sera as the growth of mammalian cells in defined media is difficult or impossible in most cases. As a result, desired protein products secreted by the cells into the medium are accompanied by large amounts of undesired serum proteins, and recovery in pure form of the desired product requires separation from these contaminants. Typically, the immunoglobulin molecules produced by hybridomas are present in the supernatants in the ug/ml range, whereas undesired proteins are present in approximately 1,000-fold greater concentrations. The availability of efficient tissue culture techniques has made possible the production of large quantities of antibodies on a scale involving volumes of hundreds or thousands of liters of culture medium. Techniques for recovery of the immunoglobulins on the mg-kg scale must take account of the necessity to deal with these large volumes, as well as the need to purify these materials away from serum proteins present in the nutrients.
A popular method for purification of some immunoglobulins from culture media has been the use of affinity chromatography, particularly affinity chromatography using protein-A, which has a general specific affinity for F.sub.c chains of certain IgG immunoglobulins. However, although this method is successful in achieving many-fold purification of the desired antibodies, the contaminants resulting directly from the materials used in the purification have been shown to have antigenic effects, and even when small in amount, they are potent in undesired bioactivity. Other disadvantages of the use of protein-A-based purification include the requirement for extreme pH conditions, the difficulty in "sanitizing" the adsorbent due to the lability of the protein-A, the inability of protein-A to bind certain IgG classes, and its failure to differentiate the desired antibody from the serum Ig in the medium. Also, if the procedure is used to purify antibodies for pharmaceutical use, the stipulation that the same resin cannot be reused for other product proteins is costly, due to the inherent cost of protein-A.
Typical methods for purifying immunoglobulins and their derivatives from cell culture medium include precipitation with salts followed by dialysis and anion exchange chromatography (Deutsch, H. F., et al, Meth Immunol Immunochem (1967), Vol. I, Academic Press, NY, p. 315); and high performance liquid chromatography (HPLC), Gemdic, M. J., et al, Biotechniques (1985) 3:378; Juarez-Salinas, H. S., ibid (1984) 2:164; Clezardin, P., J Chromatog (1985) 319:67. Affinity chromatographic methods have been based on protein-A (Hjelm, H. K., FEBS Lett (1972) 28:73) and on anti-IgG antibodies (Yelton, D. C., et al, Hybridoma (1981) 1:5). The combination of gel filtration with cation exchange chromatography is discussed in Pharmacia's customer publication Separation News (1986) vol 13.5. All of these methods, while perhaps satisfactory on a laboratory scale, are totally unsuited to scaling to a high volume procedure for the production of kilogram levels of clinical grade antibodies.
The methods of the present invention are capable of providing immunoglobulins of &gt;95% purity (wherein any remaining contaminants are benign) in a yield range of 40-75%, and in practical kilogram quantities from large volumes of culture supernatants. All of the processes employ direct contact of the concentrated or pH-adjusted medium with cation exchange resin under conditions wherein the important contaminants are not adsorbed.